Spotted fever group (SFG) Rickettsia, are intracellular pathogens that manipulate host cytoskeletal proteins to induce actin-tail formation, facilitating intracellular spread. The organization of actin-filaments in Rickettsia tails is unique compared to other motile intracellular pathogens, suggesting that Rickettsia utilize a novel mechanism of actin-tail assembly. The Rickettsia protein, RickA, is thought to play an essential role in actin tail formation. RickA is a member of the Wiskott Aldrich Syndrome Protein (WASP) family of actin nucleation promoting factors (NPF). NPFs promote actin nucleation by activation of the Arp2/3 complex, which is the initial step for all Arp2/3-dependent actin polymerization events. The long-term objectives of this research application are to define the molecular mechanism of Rickettsia actin-based motility. In order for RickA to have a direct role in actin-tail formation, the protein should be located extracellularly, and there is some evidence to suggest this. However, it is unknown whether the protein is found in the eukaryotic cell cytoplasm or if it is tethered to the bacterial membrane. Moreover, the mechanism of RickA secretion is unknown, but Rickettsia encodes a Type IV Secretion System (TFSS) and RickA contains a putative TFSS signal. Therefore, the first specific aim of this application is to determine the localization of RickA and the method by which this protein is secreted. Biochemical fractionation and immunofluorescence microscopy will be performed to address the question of RickA localization. To determine whether RickA is translocated by the TFSS, RickA will be expressed in Legionella pneumophila and its ability to be translocated by the L. pneumophila TFSS will be analyzed by immunofluorescence microscopy. The second specific aim is designed to address the role of eukaryotic factors in Rickettsia actin tail formation. RickA promotes Arp2/3-dependent nucleation in vitro, however a requirement for Arp2/3 in vivo has not yet been described. In addition, the distinct organization of Rickettsia actin filaments suggests that eukaryotic proteins important for actin organizing, such as filamin and profilin, may also be required for tail formation. Expression of these cytoskeletal proteins will be silenced by targeted RNA interference and actin tails will be analyzed by immunofluorescence microscopy. In order to provide proper protection and treatment for spotted fever diseases, it is critical to understand the basic biology behind the pathogenesis of Rickettsia. By elucidating the role of the RickA protein in Rickettsia motility, we will learn more about how human cases of infection should be treated. In addition, through understanding and identifying human cytoskeleton proteins important in the formation of the Rickettsia motile structures, a greater understanding of the human cell cytoskeleton will result. [unreadable] [unreadable] [unreadable]